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WO1999006535A1 - Dosages de criblage pour agonistes et antagonistes de recepteurs couples aux proteines g - Google Patents

Dosages de criblage pour agonistes et antagonistes de recepteurs couples aux proteines g Download PDF

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Publication number
WO1999006535A1
WO1999006535A1 PCT/US1998/015877 US9815877W WO9906535A1 WO 1999006535 A1 WO1999006535 A1 WO 1999006535A1 US 9815877 W US9815877 W US 9815877W WO 9906535 A1 WO9906535 A1 WO 9906535A1
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cells
hpth
hkrk
pth
pthr
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PCT/US1998/015877
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English (en)
Inventor
F. Richard Bringhurst
Hisashi Takasu
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The General Hospital Corporation
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Priority to DE69829483T priority Critical patent/DE69829483T2/de
Priority to AT98938160T priority patent/ATE291615T1/de
Priority to EP98938160A priority patent/EP1012241B1/fr
Priority to AU86750/98A priority patent/AU8675098A/en
Priority to JP2000505277A priority patent/JP4261766B2/ja
Publication of WO1999006535A1 publication Critical patent/WO1999006535A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to G protein coupled receptors. More specifically, screening assays for Gs and Gq protein coupled receptor agonists and antagonists are provided. Also provided are stably transfected cell lines.
  • PTH Parathyroid hormone
  • PTHrP Parathyroid hormone
  • Exogenously administered PTH exerts striking effects upon bone mass in vivo, the nature of which depends critically upon the dose of
  • PTH analogs as anabolic agents to increase bone mass and to prevent or treat metabolic bone diseases, including osteoporosis (Dempster, D.W., et al, Endocrine Rev 14:690-109 (1993), Whitfield, J.F., and Morley, P., Trends Pharmacol Sci 7(5:382-386 (1995)).
  • PTH and PTHrP can activate a single receptor, the PTH/PTHrP receptor (PTHR), which has been cloned from several species, including rat, opossum, mouse, pig and human, and shown to be expressed in cells of bone
  • PTHR PTH/PTHrP receptor
  • Activation of the PTHR in osteoblasts evokes multiple parallel signaling events, including activation of adenylyl cyclase (AC), phospholipase C (PLC) and cytosolic free calcium transients (Abou-Samra, A.B., et al, Proc Natl Acad Sci USA 89:2132-2136 (1992), Juppner, H., et al, Science 254:1024-1026 (1991), Bringhurst, F.R., et al, Endocrinology 732:2090-2098 (1993), Dunlay, R., and Hruska, K., Am J Physiol 25 ⁇ °:F223-231 (1990); Fujimori, A., et al,
  • the human PTHR has been expressed previously in cultured cells (Pines, M., etal, Endocrinology 735: 1713-1716 (1994), Schneider, H., etal, FEBSLett
  • PTH(1-31) have been previously found to exhibit selective activation of only a subset of the usual PTHR second messengers (Rixon, R.H., et al, J Bone Miner Res 9: 1179-1189 (1994), Whitfield, J.F., and Morley, P., Trends Pharmacol Sci 76:382-386 (1995), Fujimori, A., et al, Endocrinology 128:3032-3039 (1991), Abou-Samra, A.B., et al, Endocrinology 735:2588-2594 (1994); Azarani, A., et al, JBiol Chem 277: 14931-14936 (1996); Chakravarthy, B.R., et al, Biochem Beefiest Res Commun 777 : 1105- 1110 ( 1990); Fujimori, A.
  • the present inventors isolated and characterized numerous subclones of the well-characterized renal epithelial LLC-PKl cell line that collectively expressed a broad range of stable transfected human PTHRs. It was found that, as with the rat PTHR, the human receptor activates AC maximally at levels of receptor expression far lower than those needed for PLC activation. Further, the temporal pattern and magnitude of PLC activation in these cells is strongly dependent upon the density of cell-surface human PTHRs across a range of expression above that which elicits maximal AC activation.
  • hPTH(l-31) and hPTH(l-34) activate PLC and cytosolic free calcium transients equivalently via the human PTHR and, moreover, that hPTH(l -31) fully induces other, more delayed biologic responses to PTH in these cells that depend upon cAMP -independent signaling pathways.
  • the present inventors also have developed a convenient and sensitive spectrophotometric bioassay that responds to activation of either AC or PLC via the human PTHR in these cells, and have employed it, together with other measurements, to show that analogs such as PTH(3-34) and PTH(1-31), previously found to be signal- selective agonists in rat cells, may exhibit different spectra of biologic activities via the human PTHR.
  • the present invention provides a stably transfected cell line comprising LLC-PKl cells which express PTHR.
  • the present invention also provides a method for determining whether a compound of interest is an agonist or antagonist of a Gs or Gq protein coupled receptor comprising:
  • u-PA urokinase-type plasminogen activator
  • step (b) providing an expression vector comprising a nucleotide sequence encoding for a Gs or Gq protein coupled receptor, said receptor not normally expressed in said cell line of step (a);
  • step (e) measuring the u-PA activity of the cell culture supernatant of said cells of step (d).
  • the present invention further comprises a method of determining whether a compound of interest is an agonist or antagonist of a Gs or Gq coupled receptor using LLC-PKl cells in the above-described method.
  • the present invention further provides a method of determining whether a compound of interest is an agonist or antagonist of human PTHR using the above-described method.
  • FIGS 1A-1B Competitive radioligand binding to human PTH PTHrp receptors stably expressed in LLC-PKl cells. LLC-PKl cells stably expressing (Figure 1 A) 950,000 receptors/cell (HKRK B7) or ( Figure IB) 280,000 receptors/cell (HKRK B28) were incubated with
  • Figures 2A-2B Stimulation of cAMP accumulation in LLC-PKl cells expressing human PTH/PTHrP receptors. Cyclic AMP was measured in acid extracts of cells prepared after addition of the indicated peptides and incubation at 37°C for 20 min.
  • Figure 2A shows the responses to hPTH(l-34), at the indicated concentrations, in HKRK B7 cells (•; 950,000 receptors per cell) and
  • HKRK B28 cells (o; 280,000 receptors per cell).
  • Figure 2B shows the responses in HKRK B28 cells to: hPTH(l-34) (o), hPTH(l-31) ( ⁇ ), hPTH(3-34) ( ⁇ ), hPTH(7-34) ( ⁇ ), hPTHrp(l-36) ( ⁇ ), and sCT ( ⁇ ). Results were expressed as fold basal. Each point represents the mean ⁇ SEM of triplicate determinations.
  • IP 3 production expressed as percentage of basal
  • LLC-PKl cells expressing different densities of human ( ⁇ ) and rat ( ⁇ ) PTH/PTHrp receptors.
  • Cells were stimulated for 30 min with hPTH(l-34) at a concentration of 1000 nM, which elicits maximal activation of this response (see
  • FIG. 4B Each point depicts the mean ⁇ SEM of three experiments performed in triplicate .
  • the relation b etween PLC activity and receptor expression is shown schematically by the dashed lines for the rat (left) and human (right) receptors.
  • Figures 4A-4B Comparison of time-and dose dependence of IP 3 production between HKRK B7 and HKRK B28 cells.
  • Figure 4A shoes HKRK B7 cells (•) and HKRK B28 cells (o) that were stimulated with hPTH(l-34)
  • FIG. 4B shows cells that were stimulated with hPTH(l-34) at the indicated concentrations for 30 min (HKRK B7) or 4 min (HKRK B28). Results are expressed as percent of basal, and each point is the mean ⁇ SEM of three experiments performed in triplicate.
  • Basal cytosolic free calcium concentrations and those following addition of sCT in these cell lines were virtually identical - i.e. 20-50 nM and 400-500 nM, respectively. These results shown are representative of at least 5 independent experiments with each cell line.
  • FIGS 6A-6B Stimulation ofurokinase-typeplasminogen activator
  • Urokinase-type plasminogen activator activity was measured in conditioned medium of ( Figure 6A) HKRK B7 cells or ( Figure 6B) HKRK B28 cells, 16 hr after addition of agonists as follows: hPTH(l-34) (concentration shown in nM), 8BrcAMP (ImM), TPA (100 nM), or sCT (1000 nM). Results are expressed in Ploug units/well using purified human urokinase as a standard. Each bar depicts the mean ⁇ SEM of a representative experiment performed in triplicate. Similar results were obtained in over 10 individual experiments. Figure 7.
  • FIG. 8 Regulation of phosphate uptake by PTH in HKRK B7 and HKRKB28 cells. Phosphate uptake was measured in HKRK B7 cells (closed bars) and HKRK B28 cells (open bars) following incubation for 6 hr in serum-free medium containing vehicle, hPTH PTHrp peptides (1000 nM), 8BrcAMP (ImM), TPA (100 nM), or sCT (1000 nM). Preliminary experiments (not shown) demonstrated maximal responses to hPTH/PTHrp peptides at concentrations of 1000 nM or above. Results are expressed as percentage of basal, and each bar represents the mean ⁇ SEM of a representative experiment performed in triplicate.
  • FIG. 9 Stimulation of u-PA secretion by hPTH(l-31) and hPTH(l-34) in HKRK B7 cells .
  • Urokinase-type PA activity was measured 16 hr after addition of hPTH(l-34) (•) or hPTH(l-31) ( ⁇ ) at the indicated concentrations and expressed as Ploug units/well using purified human urokinase as a standard. Each point is the mean ⁇ SEM of a representative experiment performed in triplicate . Similar results were obtained in at least 6 individual experiments.
  • FIG. 10 Stimulation of cyclic AMP accumulation by hPTH(l-31) and hPTH(l-34) in LLC-PKl cells that express rat PTHRs. Cyclic AMP accumulation was measured in response to hPTH(l-34) (•) or hPTH(l-31) ( ⁇ ) in EW5 cells that expressed 320,000 rat PTHRs per cell. Results are expressed as fold basal.
  • the present inventors have developed LLC-PKl cell lines which express human PTHR.
  • the present studies of clonal LLC-PKl cell lines that collectively span a broad range of expression of stably transfected human PTHRs have demonstrated receptor density-dependent differences in hPTH signaling and biologic activity that indicate an important role for regulation of human receptor expression in modulating the character, as well as the magnitude, of the cellular response to the hormone.
  • a stably transfected cell line comprising LLC-PKl cells which express human PTHR.
  • a cell line is a population of cells of the same type that is capable of indefinite survival in culture.
  • stably transfected cell line it is meant that the cell line has been altered in some way to express a polypeptide which it does not normally express.
  • express it is meant that a structural gene is transcribed into mRNA and that such mRNA is translated to produce a polypeptide.
  • LLC-PKl cells are porcine renal epithelial cells which express calcitonin and vasopressin receptors, but do not normally express PTHR and are available from the American Type Culture Collection, ATCC No. CL-101.
  • the human PTHR is a receptor which binds to both human PTH and human PTHrP.
  • the human PTHR has been previously cloned (Schneider etal, Eur. J. Pharmacol. 246:149-155 (1993); Adams et /.,
  • LLC-PKl cells which express human PTHR are said to be "stably transfected.”
  • u-PA is secreted by LLC-PKl cells in response to activation of both the PKA and PKC pathways by calcitonin (Jans, D.A., and Hemmings, B.A., FEBS Lett 205: 127-131 (1986)).
  • the present inventors have reconfirmed that, in LLC-PKl cells, both the PKA and PKC pathways are linked to u-PA production.
  • the present inventors have developed a spectrophotometric bioassay that measures u-PA production, and thus PKA or PKC activation, in these cells. Gs and Gq proteins activate the PKC pathway and thereby increase u-PA production in LLC-PKl cells.
  • An agonist of a Gs or Gq protein coupled receptor increases u-PA production in cells which express u-PA.
  • An antagonist of a Gs or Gq protein coupled receptor inhibits the activity of a Gs or Gq protein coupled receptor agonist, thereby decreasing u-PA production in cells which express u-PA relative to the agonist administered alone.
  • Cell lines other than LLC-PKl express u-PA. These cell lines could be used to determine whether a compound is an agonist or antagonist of a Gs or Gq protein coupled receptor using the method of the present invention. Examples of cells lines which express u-PA include, but are not limited to, HE-LU (Rifkin) (ATCC No. CRL-7717); LLC-MK2 (ATCC No. CCL-7); NMU (ATCC No.
  • LLC-RK1 ATCC No. CCL-106
  • MIA PaCa-2 CRL-1420
  • a further embodiment of the present invention involves a method of determining whether a compound of interest is an agonist or antagonist of a Gs or
  • Gq protein coupled receptor comprising: (a) providing a cell line which expresses urokinase-type plasminogen activator (u-PA); (b) providing an expression vector comprising a nucleotide sequence encoding for a Gs or Gq protein coupled receptor, said receptor not normally expressed in said cell line of step (a);(c) introducing said expression vector into said cell line, thereby providing stably transfected cells; (d) contacting said stably transfected cells with said compound of interest; and (e) measuring the u-PA activity of the cell culture supernatant of said cells of step (d).
  • u-PA urokinase-type plasminogen activator
  • a preferred embodiment of the present invention is a method of determining whether a compound of interest is an agonist or antagonist of a Gs protein coupled receptor, using the above-described method.
  • Another preferred embodiment of the present invention is a method of determining whether a compound of interest is an agonist or antagonist of a Gq protein coupled receptor, using the above-described method.
  • An especially preferred embodiment of the present invention is a method of determining whether a compound of interest is a Gs or Gq protein coupled receptor agonist or antagonist using LLC-PKl cells in the above-described method.
  • Another especially preferred embodiment of the present invention is a method of determining whether a compound of interest is an agonist or antagonist of human PTHR using the above-described method.
  • a "compound of interest” could be a peptide, a polypeptide, a fragment of a polypeptide, an organic natural molecule, or a synthetic molecule.
  • Examples of compounds that could be agonists or antagonists of Gs or Gq protein coupled receptors are hormones, hormone analogs, and antibodies.
  • An "agonist of a Gs or Gq protein coupled receptor” is a compound that interacts with a Gs or Gq protein coupled receptor and activates the Gs or Gq protein coupled receptor.
  • An "antagonist of a Gs or Gq protein coupled receptor” is a compound that inhibits the agonist-induced activation of a Gs or Gq protein coupled receptor.
  • Gs or Gq protein coupled receptor is a receptor that, when bound to its appropriate ligand, activates a Gs or Gq protein. Some receptors can activate both Gs and Gq proteins, while some activate only either Gs or Gq proteins. Preferably, the Gs or Gq protein coupled receptors used in the present invention should be capable of activating Gs or Gq proteins of the cells used in the method of the present invention. Examples of Gs protein coupled receptors include, but are not limited to, the ⁇ -adrenergic, glucagon, ADH, FSH, LH, and VIP receptors. Examples of Gq protein coupled receptors include, but are not limited to, the TRH, thrombin, and PGF 2 ⁇ receptors. One example of a receptor which activates both Gs and Gq proteins is the calcitonin receptor.
  • an "expression vector” is a vector comprising a structural gene operably linked to an expression control sequence so that the structural gene can be expressed when the expression vector is stably transfected into an appropriate host cell.
  • Two DNA sequences are said to be “operably linked” if the nature of the linkage between the two nucleic acid molecules does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired sequence, or (3) interfere with the ability of the desired sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a desired nucleic acid sequence if the promoter were capable of effecting transcription of that nucleic acid sequence.
  • Preferred promoters include the promoter of the mouse metallotionein I gene (Hamer, D. et al. , J. Mol. Appl. Gen. 7:273-288 (1982)), the HSV thymidine kinase promoter (McKnight, S. Cell 37:355-365 (1982)) or the SV40 early promoter (Benoist, C. et al, Nature 290:304-310 (1981)).
  • Enhancers are cis-acting elements of DNA, generally about 10 to 300 bp in size, that act to increase transcriptional activity of a promoter.
  • Illustrative examples of enhancers include, but are not limited to, the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270; the cytomegalovirus early promoter enhancer; the polyoma enhancer on the late side of the replication origin; and adenovirus enhancers.
  • the expression vectors may provide for inducible expression of the nucleic acid sequence.
  • Preferred among such vectors are vectors which provide for expression that is inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • the expression vectors may also contain a selectable marker for propagation in stably transfected cells.
  • selectable markers include dihydrofolate reductase, hygromycin or neomycin resistance.
  • An expression vector can be provided commercially or it can be constructed through any method of cloning well-known in the art.
  • Preferred expression vectors include, but are not limited to, pWLNEO, pSV2CAT, pOG44, pXTl, and pSG available from Stratagene; psVK3, pBPV, pMSG and pSVL available from Pharmacia; and pcDNAIneo available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
  • the expression vector can be introduced into the host cell by any appropriate method, including infection, transduction, transfection, transvection, electroporation, and transformation. Such methods are described in many standard laboratory manuals, such as Davis et al. , Basic Methods in Molecular
  • not normally expressed in the cell line it is meant that the Gs or Gq protein coupled receptor is not expressed at a detectable level in the cell line used in the method of the present invention
  • contacting stably transfected cells with a compound of interest it is meant that the compound of interest is administered to the stably transfected cells by any appropriate method This can include administering the compound exogenously in vitro or in vivo, or further transfecting the cells to express the compound
  • a carrier may be used The carrier should be compatible with cell viability Examples of carriers include, but are not limited to, DMSO, MeOH/EtOH, water, T ⁇ s, and HEPES
  • the compound of interest may be highly purified, partially purified, or unpurified Compounds of interest may be administered to the stably transfected cells alone or in combination By administering the compounds in combination, synergistic effects on the Gs or Gq protein coupled receptor may be determined
  • “measuring the u-PA activity” it is intended qualitatively or quantitatively measuring or estimating the level of u-PA activity either directly (1 e , by determining or estimating absolute u-PA activity) or relatively (l e , by comparing the u-PA activity of the stably transfected cell line that has been contacted with a compound of interest to a control stably transfected cell line that has not been contacted with the compound of interest)
  • the u-PA activity m the stably transfected cell line that has been contacted with a compound of interest will be compared to the u-PA activity of either the stably transfected cell line which has not been contacted with the compound of interest or an untransfected cell line which has
  • Example 3 One preferred method of measuring u-PA activity is described in Example 3 below Briefly, an aliquot of supernatant from stably transfected cells is transferred to a clean microplate A " supernatant" is the liquid medium which has been removed from the cells A buffer for assaying u-PA activity is added The microplate is incubated for an appropriate amount of time, and the reactions are stopped with a termination buffer. Absorbance of the colorimetric product is measured.
  • the compound when determining whether a compound of interest is an agonist, is administered to stably transfected cells and level of u-PA activity is measured or estimated and compared to the level of u-PA activity in stably transfected cells which have not come in contact with the compound of interest.
  • both the compound of interest and a known agonist should be administered to the stably transfected cells.
  • the agonist and potential antagonist can be administered in any order (ie., agonist first and then potential antagonist or potential antagonist first and then agonist), as well as concurrently.
  • the level of u-PA activity in the stably transfected cells is measured or estimated and compared to the level of u-PA activity in stably transfected cells which have been contacted with the agonist alone.
  • the cloned porcine kidney-derived cell line, LLC-PKl (Bringhurst, F.R., et al, Endocrinology 732:2090-2098 (1993)) and subclones isolated after stable transfection with human PTH/PTHrp receptor cDNA were maintained in DMEM supplemented with 7% FB S and 1 % penicillin/streptomycin with or without 1000 ug ml G418 (all from GIBCO-BRL, Grand Island, NY) under 5% CO 2 in air.
  • the binding reaction was terminated by aspirating the incubation mixture, after which the cells were washed twice with 0.5 ml of ice-cold Buffer A. After solubilizing the cells with 0.5 ml of Lysis Buffer (0.5 N NaOH + 0.1% Triton X-100), measurements of radioactivity and protein were performed to calculate the receptor number per cell by Scatchard analysis, as previously described (Bringhurst, F.R., et al, Endocrinology 732:2090-2098 (1993)). All reagents, unless otherwise specified, were obtained from Sigma (St. Louis, MO), and all isotopes were purchased from Dupont-New England Nuclear (Boston,MA).
  • Radioligand competition assays and Scatchard analyses for two representative cell lines, HKRK B7 and HKRK B28, which express 950,000 and 280,000 hPTHRs per cell, respectively, are shown in Figure 1.
  • Scatchard analysis of the all of the selected cell lines demonstrated a range of PTHR expression from
  • Cellular cAMP accumulation Cells were seeded into 24-well plates at a density of 2.5x10 cells/well and cultured for a further 2 days before study. The cells were rinsed once with 0.5 ml of ice-cold Buffer B [10 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM KC1, 1.2 mM CaCl 2 , 1 mM MgCl 2 , 1.2 mM 5 mM glucose, and 0.1% heat-inactivated BSA] supplemented with 1 mM isobtylmethylxanthine (IBMX), and placed on ice. Treatments were added to each well in 0.25 ml of Buffer B [10 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM KC1, 1.2 mM CaCl 2 , 1 mM MgCl 2 , 1.2 mM 5 mM glucose, and 0.1% heat-inactivated BSA] supplement
  • Cells were seeded into 24-well plates at a density of 2.5x10 cells/well and cultured for a further 2 days before study. The cells were labeled at 37°C for
  • IP 3 fractions were collected and their content of radioactivity was determined by liquid scintillation spectrometry (Beckman, model LS 6000IC).
  • Cytosolic free calcium was measured by dual fluorescence in cells loaded with the Ca -sensitive intracellular probe fura-2.
  • Cells were seeded onto glass coverslips at a density of 40,000-600,000/cm and incubated for 2 days as described above. Coverslips were washed twice with PBS before loading in phosphate-free Buffer B containing 4 uM of fura-2/ AM (Molecular Probes,
  • Peptide stocks were prepared in 0.1% trifluoroacetic acid, ionomycin and phorbol esters were dissolved in dimethyl sulfoxide and all other additives were prepared in water. Maximum and minimum fluorescence signals were obtained by exposure to modified buffers containing 1 uM ionomycin plus 20 mM calcium or 2 mM EGTA in the nominal absence of calcium, respectively, and autofluorescence was estimated from signals obtained in the presence of ionomycin plus 2.5 mM MnCl 2 . Cytosolic free calcium then was determined as previously described (Bringhurst, F.R., et al, Endocrinology 732:2090-2098 (1993)).
  • Urokinase-type plasminogen activator Cells were seeded into 96-well plates at a density of 6x10 cells/well and used the following day. The cells were washed once with 0.2 ml and then refed with 0.1 ml of prewarmed DMEM containing 0.05% BSA. After adding each stimulator, plates were returned to the incubator at 37° C for 16 h. Conditioned medium (5 ul) then was transferred from each well to a clean microplate. Reactions were initiated by addition of 50 ul of u-PA assay buffer [90mM
  • Tris-HCl pH 8.8
  • Triton X-100 14 ug/ml human plasminogen (Calbiochem, San Diego, CA) and 0.4 mg/ml S-2251 plasmin substrate (D-Val-Leu-L-Lys-NH-Np) (Sigma, St. Louis, MO)].
  • Plates were incubated first at 37 °C for 15 min and then at room temperature (22 °C) for 30 min. The reactions were terminated by addition of 10 ul of ice-cold 25% acetic acid.
  • DMEM + 0.1% BSA serum-free medium
  • PTHR density among LLC-PKl subclones expressing human PTHRs As in the case of AC activation, the relative efficiency with which the human PTHR coupled to PLC in these cells was approximately 3 -5-fold less than that of the rat receptor. - i.e., roughly 3 -5-fold more human than rat receptors were required for equivalent activation of PLC.
  • PTH-responsive cell lines that express lower numbers of PTHRs - i.e. UMR 106 cells (approximately 70,000 receptors per cell) - a pattern of very rapid but transient PLC activation has been described (Yamaguchi, D.T., et al, JBiol Chem 262:11 ⁇ ⁇ -11 ⁇ (1987)). Seeking to determine if such a transient pattern might underlie the absence of a sustained PLC activation in
  • LLC-PK 1 cells expressing fewer than 400,000 human PTHRs preliminary studies were performed that demonstrated increased IP 3 at an earlier time (4 min) in HKRK C53, HKRK B28 and HKRK C101 cells (120,000, 280,000 and 330,000 PTHRs per cell, respectively), though not in HKRK B64 cells (90,000 sites per cell) (data not shown).
  • the time course of PLC activation then was studied in more detail in HKRK B28 cells and compared with that in HKRK B7 cells.
  • transient PTH-stimulated IP 3 formation was observed in HKRK B28 cells within the first few minutes of PTH exposure. This response peaked at 4 min and was followed by a rapid decrease to basal levels by 10 min.
  • IP 3 formation in HKRK B7 cells continued to increase between 10 and 30 minutes, although a more rapid initial phase also was evident in these cells.
  • HKRK B7 cells and HKRK B28 cells previously loaded with [ 3 H]myo-inositol, were incubated with each peptide (1000 nM) for 30 min or 4 min, respectively.
  • the formation of IP 3 is expressed as a percentage of the amount measured in vehicle-treated controls. Values shown are means + SEM of triplicate determinations.
  • control 100 ⁇ 5 100 ⁇ 3 hPTH (1-34) 298 ⁇ 24 172 ⁇ 9 hPTH (l-31) 275 ⁇ 21 170 ⁇ 14 hPTH (3-34) 103 ⁇ 1 85 ⁇ 9 hPTH (7-34) 122 ⁇ 7 92 ⁇ 10 hPTHrP (1-36) 93 ⁇ 6 160 ⁇ 9
  • AB45 cells were studied, in which human PTHRs (370, 000/cell) were co-expressed with REV AB, a dominant-negative inhibitor of both basal and hormone-stimulated protein kinase A (PKA) (Clegg, C.H., et al, J Biol Chem 262: 13111-13119 (1987), Fukayama, S., etal, Endocrinology 734:1851-1858 (1994)).
  • PKA protein kinase A
  • u-PA urokinase-type plasminogen activator
  • the sensitivities and magnitudes of the cAMP responses to PTH in these two cell lines were identical, it seemed possible that the greater maximal u-PA response to PTH observed in HKRK B7 versus HKRK B28 cells might reflect the sustained activation of PLC seen only in the HKRK B7 cells.
  • the EC 50 for stimulation of u-PA production by hPTH(l-34) (10-20 nM) was more similar to that for activation of IP 3 formation (20-30 nM) than for cAMP accumulation (1 nM), particularly in HKRK B7 cells.
  • the maximal u-PA response to PTH was higher than that of 8BrcAMP in HKRK B7 (but not in HKRK B28 cells), consistent with activation, by the more abundant hPTHRs in these cells, of an additional, PKA-independent mechanism. Further evidence for involvement of a cAMP-independent (presumably PKC-dependent) mechanism of PTH-induced u-PA secretion in these cells was obtained using the cAMP-resistant AB45 cells ( Figure 7). As expected, the u-PA response to 8BrcAMP was nearly obliterated in these cells, whereas that to phorbol ester was well-maintained.
  • the present inventors report similarly striking differences in sensitivity of the AC and PLC responses to PTH in LLC-PKl cells that express hPTHRs - i.e. the EC 50 that was observed for activation of AC was 10- 100-fold lower than that for PLC stimulation.
  • This "rightward shift" of the relation between overall signaling efficiency and receptor density may reflect an intrinsic property of the human PTHR or, alternatively, a relative species incompatibility between the human receptor and the intracellular signal transducers expressed by the porcine cells used here. More efficient coupling of transfected human PTHRs to PLC or cytosolic calcium signaling has been observed in cells of primate or human origin (Pines, M., et al, Bone 75:381-389 (1996), Schipani, E., etal, Science 265:98-100 (1995)). Direct comparisons of the functions of transfected rat and human PTHRs in cells derived from a wider range of species will be needed to clarify this issue.
  • HKRK B7 cells could be due simply to a more intense and protracted activation of the enzyme, sustained by a much larger number of occupied receptors held in an active configuration in these cells, that was sufficient to nearly saturate the phosphatases involved in metabolizing IP 3 (Shears, S.B., Biochem J 260:313-324 (1989)).
  • the results seem most consistent with this possibility, as no difference could be detected in EC 50 's of the responses measured at 4 min vs. 30 min, nor was an obvious difference detected in ligand selectivity that might have pointed to two different mechanisms.
  • activation of AC by PTH appeared to proceed identically over the first 15 min in both cell lines.
  • a third interesting feature of hPTHR signaling in these cells concerns the cytosolic calcium responses that were observed.
  • IP 3 is a major determinant of calcium release from intracellular stores during activation of G protein-coupled receptors (Berridge, M.
  • PTH analogs were reported to exhibit signal-selectivity in PTH-responsive cells of non-human origin. These include PTH(3-34) and PTH(7-34), which have been shown in some systems to selectively activate PKC and/or cytosolic calcium transients (Fujimori, A., et al, Endocrinology 725:3032-3039 (1991), Abou-Samra, A.B., etal, Endocrinology 735:2588-2594
  • hPTH(3 -34), hPTH(7-34) and hPTH( 1-31) were concordant with their signaling properties.
  • hPTH(3-34) was partially active (50-60%) of maximal response to hPTH(l-34))
  • hPTH(7-34) was inactive and hPTH(l-31) was fully active (Table 3, Figure 9).
  • hPTH(l-31) and hPTH(l-34) showed comparable maximal activation of phosphate uptake, which is PKC-dependent in these cells (Guo, J., etal, Endocrinology 736:3884-3891 (1995)), whereas hPTH(3-34) and hPTH(7-34) were inactive, as expected ( Figure 8).
  • hPTH(l-31) Stimulation of phosphate uptake by the two peptides also was identical.
  • the ability of hPTH(l-31) to activate PLC and other cAMP -independent responses via the PTHR was not restricted to the human receptor but was seen also in LLC-PKl cells that express recombinant rat PTHRs.
  • the PLC response to hPTH( 1-31) may be modestly impaired, relative to hPTH(l-34), via the rat PTHR, whereas the human receptor seems unable to discriminate between these two ligands.
  • hPTH(l-34) or hPTH(l-31) were measured in EW5 cells, which express 320,000 rat PTHRs per cell. Each value is the mean ⁇ SEM of a representative experiment performed in triplicate. Similar results were obtained in at least 3 individual experiments. The modest reduction in IP 3 formation observed with hPTH(l-31) relative to hPTH(l-34) was confirmed in 3 other LLC-PKl cell lines that expressed rat PTHRs (i.e.
  • EW29, AR-C38 and AR-B44 which express 190,000, 630,000 and 800,000 PTHRs/cell, respectively), in which maximal IP 3 formation after hPTH(l-31) (as % of hPTH(l-34) effect) was 75%, 77% and 64%, respectively.
  • hPTH(l-31) was fully equivalent to hPTH(l-34) with respect to activation of AC, PLC, cytosolic calcium transients, u-PA secretion and phosphate uptake in LLC-PKl cells that express hPTHRs.
  • hPTH(l-31) had been reported to retain AC activity but to be devoid ofPKC-stimulating activity (Rixon, R.H., et al, J 'Bone Miner Res 9: 1179-1189 (1994), Jouisans, H., et al, J Bone Miner Res 9:943-949 (1994), Neugebauer, W., etal, Biochemistry 34:8835-8842 (1995)).
  • ROS 17/2 cells almost certainly express the same rPTHR that were studied here, as they were the source of the ROS 17/2 8 subclone from which the rPTHR cDNA originally was cloned and then used to create the rPTHR-expressing LLC-PKl cells used in the experiments (Abou-Samra, A.B., et al, Proc Natl Acad Sci USA 59:2732-2736 (1992), Bringhurst, F.R., et al, Endocrinology 732:2090-2098 (1993), Guo, J., et al, Endocrinology 736:3884-3891 (1995)).
  • ROS 17/2 cells and spleen cells express alternate species of receptors for PTH that could have influenced the response(s) to hPTH( 1 -31 ) in these cells.
  • a PTH receptor with apparent C-terminal specificity has been described in ROS 17/2.8 cells (Inomata, N., et al, Endocrinology 736:4732-4740).
  • the ROS 17/2 and rat spleen cell systems differ in other ways from the rPTHR-transfected LLC-PKl cells that were studied: (a) the surface expression of PTHRs is known, or likely, to be lower than that in the LLC-PKl cells were used, (b) rPTHRs are coupled to rat rather than porcine G proteins, and (c) the relative quantities of the various subtypes of expressed G proteins and effector enzymes could be very different. The issue of lower PTHR expression may be particularly important. The data suggest that the potency of the hPTH(l-31) peptide for PLC/PKC activation via the rat PTHR is moderately reduced (by 40%), relative to that of hPTH(l-34).

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Abstract

L'hormone parathyroïde (PTH) et le peptide PTHrP qui y est étroitement lié, partagent le même récepteur, PTHR. Les cellules LLC-PK1 sont des cellules épithéliales rénales du porc qui, normalement, n'expriment pas le PTHR. Cette invention concerne des cellules LLC-PK1 transfectées de manière stable qui expriment le récepteur PTHR humain. Cette invention concerne également des procédés permettant de déterminer si un composé est un agoniste ou un antagoniste du récepteur couplé à une protéine Gs ou Gq.
PCT/US1998/015877 1997-07-31 1998-07-30 Dosages de criblage pour agonistes et antagonistes de recepteurs couples aux proteines g WO1999006535A1 (fr)

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DE69829483T DE69829483T2 (de) 1997-07-31 1998-07-30 Testverfahren für Gq Protein gekoppelter Rezeptur-Agonisten und Antogonisten
AT98938160T ATE291615T1 (de) 1997-07-31 1998-07-30 Testverfahren für agonisten und antagonisten des g protein gekoppelten rezeptors
EP98938160A EP1012241B1 (fr) 1997-07-31 1998-07-30 Dosages de criblage pour agonistes et antagonistes de recepteurs couples aux proteines g
AU86750/98A AU8675098A (en) 1997-07-31 1998-07-30 Screening assays for g protein coupled receptor agonists and antagonists
JP2000505277A JP4261766B2 (ja) 1997-07-31 1998-07-30 Ｇタンパク質結合レセプターアゴニストおよびアンタゴニストについてのスクリーニングアッセイ

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US7166463B2 (en) * 2001-11-16 2007-01-23 The Regents Of The University Of Colorado Nucleic acids encoding modified olfactory cyclic nucleotide gated ion channels
EP1610813A4 (fr) * 2003-03-19 2009-07-01 Gen Hospital Corp Hormones parathyroidiennes contraintes de maniere conformationnelle avec des stabilisateurs en helice $g(a)
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CA2694667C (fr) * 2007-08-01 2018-10-30 The General Hospital Corporation Procedes de criblage a recepteurs couples a la proteine g et compositions apparentees
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KR101900078B1 (ko) 2010-05-13 2018-09-18 더 제너럴 하스피탈 코포레이션 부갑상선 호르몬 아날로그 및 그의 용도
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